Dapi dye

Cethyltrimethylammonium bromide (CTAB), FeCl₂.4H₂O, FeCl₃.6H₂O, HAuCl, NH₂OH.HCl, dithiotreitol (DTT), sodium citrate, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Tris buffer acetate-EDTA (TAE), dimethyl sulfoxide (DMSO) and DAPI dye were purchased from Sigma, USA. Ethidium bromide and LysoTracker Red DND-99 were obtained from Life Technologies, USA. Human breast cancer (MCF-7) and Chinese hamster ovarian (CHO) cells were purchased from Pasteur Institute, Iran. DMEM medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, USA). MUC-1 Aptamer as a targeting moiety was purchased from TAG Copenhagen A/S, Denmark. Amicon ultracentrifugal tube-Millipore (10 KDa) was purchased from Merck, Germany.

Mice were anesthetized with xylazine and ketamine as described above and had their left ventricles perfused with 4% (w/v) paraformaldehyde (PFA, Sigma, St Louis, MO, USA) in phosphate-buffered saline (PBS) (pH 7.4). The TA muscles were then carefully removed. Tissues were post-fixed in 4% PFA over 12 hours, submersed in 30% sucrose overnight, frozen in OCT embedding compound, and sectioned coronally (10 microns) with a cryostat. Cryo-sections were thaw mounted onto gelatin-coated slides and stored at -20°C.
Frozen sections, after being rinsed three times in PBS, were permeabilized and blocked with PBS containing 0.1% (v/v) Triton X-100 and 10% (v/v) goat serum in PBS for 1 hr at room temperature (RT) and then incubated with either rabbit-anti-dystrophin antibody (1:600, Abcam Biochemicals, Cambridge, MA, USA) or mouse-anti-Pax7 antibody (1:50, Hybridoma Bank, Iowa City, IA, USA). The sections were then incubated with the goat-anti-rabbit CyTm5-conjugated or goat-anti-mouse Cy3-conjugated secondary antibody (Jackson Lab) for 1 hr at RT. Slides were counterstained with DAPI dye (1:800, Sigma, St Louis, MO, USA), rinsed and coverslipped with fluorescence mounting medium (Dako, Glostrup Denmark). Stained sections were examined under the Olympus fluorescence microscope (Olympus, Center Valley, PA, USA), and digital images of sections were acquired with a CCD camera.

After fixation with paraformaldehyde and dehydration with sucrose, tissues of days 1 and 2 were embedded in OCT. 5 μm-thick section was incubated with primary antibodies against the M2 marker Arg-1 (Abcam) overnight at 4°C and then with the Alexa Fluor-conjugated IgG secondary antibodies (Santa) for 1 h at room temperature and finally followed by DAPI dye (Sigma) for 5 min. The cells were examined with a confocal imaging system (Olympus FV1200).
Moreover, the detection of the transplanted CM-Dil-labeled ASCs at day 14 was performed through immunofluorescence, and the tissues from day 14 were incubated with antibody vWF (Abcam) using the same method described above.

To determine if angiogenesis had developed around the scaffolds, immunofluorescent staining for endothelial cell markers (CD31) was performed for each group. Following overnight incubation at 4 ​°C with a rabbit polyclonal primary antibody to CD31 (Ab28364, 1:100, Abcam, USA), the samples were incubated with a goat anti-rabbit IgG H&L secondary antibody (1:50, Abcam, USA) at room temperature for 90 ​min. The nuclei were stained with DAPI dye (Sigma Aldrich, no. D9542, US). The density of capillaries was calculated by counting the number of VEGFR2-positive vascular structures in three randomly chosen fields/sections.

Coronarin D (purity > 95%) was purchased from ChemFaces (Wuhan, Hubei, China). It was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium to the final concentration on the experimental day. The final concentration of DMSO for all treatments was consistently less than 0.1%. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), RNase A, DAPI dye, protease inhibitor cocktail, phosphatase inhibitor cocktail, monodansylcadaverine (MDC), acridine orange (AO), and 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibody against cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), LC3B, p62, NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2 TSC1, TSC2, Rheb, p-mTOR (Ser2448), mTOR, p-AKT, AKT, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK1/2, JNK1/2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific inhibitors for N-Acetyl-L-cysteine (NAC) AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125) Wortmannin and Bafilomycin A1 (Baf A1), and z-VAD-FMK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).